Two-stage conversion of syngas and pyrolysis aqueous condensate into L-malate

Hybrid thermochemical–biological processes have the potential to enhance the carbon and energy recovery from organic waste. This work aimed to assess the carbon and energy recovery potential of multifunctional processes to simultaneously sequestrate syngas and detoxify pyrolysis aqueous condensate (PAC) for short-chain carboxylates production. To evaluate relevant process parameters for mixed culture co-fermentation of syngas and PAC, two identical reactors were run under mesophilic (37 °C) and thermophilic (55 °C) conditions at increasing PAC loading rates. Both the mesophilic and the thermophilic process recovered at least 50% of the energy in syngas and PAC into short-chain carboxylates. During the mesophilic syngas and PAC co-fermentation, methanogenesis was completely inhibited while acetate, ethanol and butyrate were the primary metabolites. Over 90% of the amplicon sequencing variants based on 16S rRNA were assigned to Clostridium sensu stricto 12. During the thermophilic process, on the other hand, Symbiobacteriales, Syntrophaceticus, Thermoanaerobacterium, Methanothermobacter and Methanosarcina likely played crucial roles in aromatics degradation and methanogenesis, respectively, while Moorella thermoacetica and Methanothermobacter marburgensis were the predominant carboxydotrophs in the thermophilic process. High biomass concentrations were necessary to maintain stable process operations at high PAC loads. In a second-stage reactor, Aspergillus oryzae converted acetate, propionate and butyrate from the first stage into L-malate, confirming the successful detoxification of PAC below inhibitory levels. The highest L-malate yield was 0.26 ± 2.2 molL-malate/molcarboxylates recorded for effluent from the mesophilic process at a PAC load of 4% v/v. The results highlight the potential of multifunctional reactors where anaerobic mixed cultures perform simultaneously diverse process roles, such as carbon fixation, wastewater detoxification and carboxylates intermediate production. The recovered energy in the form of intermediate carboxylates allows for their use as substrates in subsequent fermentative stages. Supplementary Information The online version contains supplementary material available at 10.1186/s13068-024-02532-2.


Fermentation medium
The modified BA medium was composed as follows.For each liter of medium added: 100 mL of mineral salt solution, 800 mL of phosphate buffer solution, 10 mL of vitamin solution, 10 mL of trace elements solution, 5 mL of resazurin solution and 3 mL of reducing agent solution.

Syngas and PAC load composition
Table S2.Composition in gCOD/Ld and electron moles (e-mM/d) of the feed (for both syngas and PAC) for both the mesophilic and the thermophilic semi-continuous fermentations.

Equations Estimation of VSS concentration
Between each re-inoculation event, the concentration of volatile suspended solids (VSS) at time t=j was determined as described by Eq. 1.
, =   −  , [g/L] Eq.1 Where TSSj was determined experimentally at time t=j.As mentioned in the materials and methods section, the weight of the dried pellet was measured and assumed to be representative of the suspended fraction (TSS) of the total solids (TS).The residual amount of the total fixed solids (TFSRef,j) was calculated using the following equation: Eq.2 TFSRes,j-1 is the residual amount of the total fixed solids determined at time t=j-1.HRT is the hydraulic retention time and was HRT = 20 d in this study.The initial TFS was determined by multiplying the TFS of the inoculum by the dilution factor of the inoculation.

PAC components removal
The concentration of each selected PAC component in the fermentation medium was determined via HPLC analysis of a solution composed of BA medium with PAC concentrations similar to those reported in Supp.Where Cm,j is the concentration of compound i at time t=j determined experimentally via HPLC and CTh.Broth,m,j is the theoretical concentration of the compound i at time t=j in the fermentation broth considering an accumulation in an abiotic system, as shown in equation Eq.4 Eq.4 Where CTh.Broth,m,j is the theoretical concentration of the compound i at time t=j-1; CFeed,m is the determined concentration of the compound i in the feed and d is the dilution rate of the system.The volume of the broth is considered constant and equal to 1.5 L and constant are considered to be also the daily feed and removal (0.075 L/d) of the fermentation broth.

E-mol balances
The daily e-mol recovery was calculated as described by Eq. 5 to determine the daily load of emol from PAC.

𝑅𝑅-𝑅𝑅𝑅𝑅𝑅𝑅 𝑅𝑅𝑅𝑅𝐸𝐸𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝐸𝐸
Eq.5 The ∑e-molSubstrates is the sum of the daily e-mol from syngas and from PAC per broth volume fed into the bioreactors.The volume of the broth is considered constant and equal to 1.5 L.
Hydrogen is considered a substrate only when it was consumed, otherwise a product and was accounted as such.
Where ̇ of CO and H2 are the daily uptake rates determined as described in another work [1].̇COD,PAC is the daily flow rate of gCOD of PAC in the feed.We assumed that 1 mol of electron equivalents is equal to 8 gCOD.Considering that the COD (Chemical oxygen demand) is the oxygen required to completely oxidize the carbonaceous fraction of organic compounds and that 1 eeq. is released upon complete oxidation of carbonaceous compounds, then from the half reaction RS1, is can assumed that 1/4 mol of O2 (8 g) would be consumed in accepting the 1 emol.
The ∑e-molProducts the sum of the daily production rate in e-mol of methane, H2 (when produced), formate, acetate, ethanol, propionate and butyrate.It was calculated as described in equation Eq.8.Negative productivities were not accounted.
- ̇, = - ,−1 *  + - , − - ,−1 Eq.8 For compounds like acetate and propionate, which have a significant concentration in the PAC, the daily acetate e-mol feed was subtracted to the value calculated with Eq.8

Figure S1 .
Figure S1.Fermentation profile of the mesophilic process.Top x-axis shows increases in PAC loading, bottom x-axis shows the elapsed fermentation time.The red bar indicate the period of weekly re-inoculations.(a) pH and redox potential.(b) Partial pressures of CO and H2.(c) Concentration of undissociated carboxylates.(d) Removal efficacy of each cresol isomer.Negative values indicate production.(e) Concentrations of total (TSS) and volatile suspended solids (VSS).

Figure S3 .
Figure S3.Spearman's rank correlations between relative abundance of methanogens (based on mcrA gene amplicon sequencing variants) and process parameters for the thermophilic semi-continuous STR enrichment.The strength of the correlation is represented by the size of the circle and intensity of the color.Blue circles indicate positive correlations.Red circles indicate negative correlations.p values are shown for non-significant correlations (p<0.05).

Figure
Figure S4.CO, H2, CH4 production rates during the decrease in CO uptake rates for the thermophilic syngas and PAC co-fermentation.

Table S3 .
Conversion factors for electron balances.